Project Summary/Abstract: The focus of this proposal is to investigate the feasibility for therapeutic intervention in Huntington?s Disease (HD) through the modification of Huntingtin (HTT) by phosphorylation which may activate its normal function as a selective autophagic scaffold, resulting in HTT degradation. HTT is phosphorylated on serine (S) 13 by the kinase IKK??and on S421 by AKT, modifications that are reduced in mutant HTT contributing to a loss of HTT function and accumulation of proteins and organelles normally cleared by autophagy, including mutant HTT itself. We now propose to carry out an integrated series of studies which seek to explore this hypothesis in depth. We hope to provide a basis for therapeutic intervention for HD through detailed understanding of the mechanisms which regulate HTT function and degradation, and distinguish mutant HTT from normal HTT. Specifically, we will investigate the following specific aims: Specific Aim 1: Test the hypothesis that IKK? signaling plays a key role in the regulation of autophagy, HTT phosphorylation and mutant HTT clearance in vivo. We have previously found that the kinase IKK? phosphorylates HTT S13 in vivo and activates its clearance, potentially modulating its function as a scaffold for selective autophagy. To understand this process we have created a conditional tamoxifen-inducible IKK? knockout in tissue including mouse liver and striatum. We will evaluate the contribution of IKK? knockout early and late in HD progression in zQ175 knockin HD mice which are a good model of the human heterozygous disease condition. A series of assays will be performed, including mouse behavior, neuropathological assessment of mouse brain and HTT protein accumulation, and analysis of the transcriptome to provide an integrated profile of IKK? and HTT phosphorylation in these mice. Specific Aim 2: Analyze a phosphorylation-regulated ubiquitin binding domain within HTT. I propose to investigate the properties and function of an ubiquitin-binding domain within HTT amino acids 1-480 to better understand the role of this domain in modulating HD pathology, as this domain may mediate interaction between ubiquitinated autophagic cargos with the HTT scaffold to regulate their clearance by the lysosome. Specific Aim 3: Define ubiquitinated protein substrates that interact with HTT?s phosphorylation- regulated ubiquitin binding domain. I will use in vivo crosslinking in cells grown under different stress conditions to couple ubiquitinated cargos to HTT?s ubiquitin-binding domain, then use tandem co- immunoprecipitation followed by mass spectrometry analysis to characterize the types of substrates that may be cleared by selective autophagy mediated by HTT, which may lead to HD therapeutic target discovery.